Antibodies directed against the capsular polysaccharides of the pathogen Streptococcus pneumoniae protect humans against infection, and are elicited by vaccination with polysaccharide or polysaccharide conjugated to protein carriers. In the proposed study the variable region gene usage and junctional diversity of human antibodies specific for S. pneumoniae capsular serotypes 6B, 14, and 23F will be determined by repertoire cloning and sequence analysis. The influence of plain polysaccharide and polysaccharide-protein conjugate vaccine formulations on the expressed repertoire will be investigated, and the degree to which these thymus- independent and thymus-dependent forms of the vaccine induce somatic mutations and affinity maturation will be determined. The structural determinants of anti-polysaccharide antibody affinity will be defined by sequence comparison, site directed mutagenesis, and molecular modeling. Sequence-defined Fab fragments will be expressed in vitro , their affinity and fine specificity determined, and the relationship between antibody affinity and protective efficacy established using an in vitro opsonophagocytosis assay. Our overall hypothesis is that the quality of an oligoclonal antibody response, such as that seen in humans to bacterial capsular polysaccharides, is influenced to a greater degree by the affinities of the individual antibody binding domains than would be a polyclonal response. These differences in antibody affinity arise as a consequence of variable region gene usage, junctional diversity, and somatic mutation. The generation of affinity loss variants by somatic mutation could therefore leads a diminution of overall antibody quality. These studies will determine if antibodies to structurally distinct polysaccharides utilize the same or distinct variable region genes and the degree to which maturation of the response through somatic mutation determines overall affinity of the response. These studies will also determine if the same clonotypes occur in unrelated individuals, and if a single clonotype predominates the response to a given specificity. Defining the relationship between binding site affinity and antibody functional quality will provide better surrogate markers of protective immunity. Understanding the molecular mechanisms that shape the human antibody repertoire to pneumococcal polysaccharides may also suggest strategies that would facilitate the development of more efficacious vaccines.